Clinical diagnosis of non-alcoholic fatty liver disease using a panel of human blood protein biomarkers

ABSTRACT

The invention relates to methods of diagnosing, prognosing, or monitoring or staging the progression of non-alcoholic fatty liver disease (NAFLD) using biomarkers. The invention also relates to a method of scoring to determine the severity of NAFLD, and a method of treating NAFLD.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/GB2017/052486, filed Aug. 23, 2017, which claims the priority to GB1614455.2, filed Aug. 24, 2016, which are entirely incorporated hereinby reference.

FIELD OF INVENTION

The invention relates to methods of diagnosing, prognosing, ormonitoring or staging the progression of non-alcoholic fatty liverdisease (NAFLD) using biomarkers. The invention also relates to a methodof scoring to determine the severity of NAFLD, and a method of treatingNAFLD.

BACKGROUND TO THE INVENTION

NAFLD is the most common liver disorder in the Western world. Itencompasses a disease spectrum of progressive liver disease ranging fromnon-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH)which can develop with hepatic fibrosis and cirrhosis. The disease isassociated with obesity and approximately 1 in 3 people in the US and UKhave some degree of NAFLD and on average about 1 in 5 people worldwide.NAFLD is underdiagnosed since patients are usually asymptomatic evenprior to the development of end-stage liver disease (ESLD).

The main stages of NAFLD comprise non-alcoholic fatty liver (NAFL),non-alcoholic steatohepatitis (NASH), hepatic fibrosis (liver fibrosis)and cirrhosis.

“Non-alcoholic fatty liver” or “NAFL” is also referred to as simplesteatosis. It is where there is an accumulation of fat (triglycerides)and other lipids in the hepatocytes of the liver. Disordered fatty acidmetabolism leads to steatosis and can be caused by various factors suchas insulin resistance in diabetes, lack of exercise and excess foodintake where there is an imbalance between calorific intake andcombustion.

“Non-alcoholic steatohepatitis” or “NASH” is a more aggressive stage ofNAFLD where there is hepatic inflammation accompanied with steatosis. InUK and Europe as many as 1 in 20 people have NASH. About 20% of patientsundergoing liver transplants are patients with NASH. Cases of NASH areincreasing in the general population including children and adolescents.

“Hepatic fibrosis” can be used interchangeably with “liver fibrosis”.Hepatic fibrosis is a wound healing response characterized by theexcessive accumulation of scar tissue (i.e. extracellular matrix) in theliver. Hepatic fibrosis may or may not be seen in NASH and is wherenormal structural elements of tissues are replaced with excessiveamounts of non-functional scar tissue. Hepatic fibrosis can be caused byvarious factors including fatty liver, alcohol and viruses. Liverfibrosis is usually staged by severity using various staging methodssuch as Ishak, Metavir, Knodell and also the Kleiner-Brunt method forNAFLD.

“Hepatic cirrhosis” or “cirrhosis” is the most severe form of liverscarring and, unlike hepatic fibrosis, is nodular and causesirreversible architectural damage to the liver. Cirrhosis is the causeof over 6000 deaths every year in the UK and approximately 27,000 in theUSA, making it the ninth leading cause of death. Cirrhosis is a majorrisk factor for hepatocellular carcinoma (HCC) and, at this stage ofliver cancer, the only curative approach is liver transplantation. It isimperative to diagnose fibrosis in the early stages of reversible liverscarring so that irreversible liver damage in cirrhosis can beprevented.

The reference standard for assessing liver fibrosis and NAFLD is theliver biopsy and this invasive procedure has a number of well-knowndisadvantages: discomfort to the patient (pain and bleeding); it can beunreliable due to fibrosis being not homogenous throughout the liver;the false negative rate can be as high as 20%; an associated mortalityrate of 1 in 10,000; inter-observer variability and high cost.Ultrasound can be used to diagnose NAFLD although the sensitivity ofthis approach is poor in clinically obese patients who are the mostlikely subjects to have NAFLD. Ultrasound also is unable to distinguishNASH and fibrosis within NAFLD and has low sensitivity for steatosis(Poynard et al., 2005, Comparative Hepatology, 4, 10; Yao et al., 2001,J Ultrasound Med, 20, 465; Schwenzer et al., 2009, J Hepatol, 51, 433).In a similar way to biopsy, ultrasound assessment of NAFLD isoperator-dependent with an inter-observer variation (Strauss et al.,2007, Am J Roentgenol; 189, W320). Magnetic resonance imaging (MRI) is apromising approach to diagnose liver fibrosis (Banerjee et al., 2014, JHepatol, 60, 69) which works for the majority of patients although itcannot be used for a very small number of patients who areclaustrophobic, unable to keep still, unable to fit into the MRI scannerand for patients with internal medical devices such as pacemakers.Transient elastography (FibroScan) assesses liver stiffness and althoughthis method is promising for liver fibrosis, it is difficult to performin obese patients which make up most NAFLD patients (Petta et al., 2011,Aliment PharmacolTher, 33, 1350).

Consequently, there is a need for improved, minimally-invasive methodsof determining stages of NAFLD in patients.

SUMMARY OF THE INVENTION

The inventors have identified protein biomarkers that are either up- ordown-regulated in patients who are at different stages of NAFLD, such asNAFL, NASH with/without fibrosis, cirrhosis.

The invention provides a method of diagnosing, prognosing or monitoringor staging the progression of non-alcoholic fatty liver disease (NAFLD)in an individual, the method comprising detecting and quantifying one ormore biomarkers in a biological sample obtained from the individual,wherein the one or more biomarkers is selected from apolipoprotein F,lipopolysaccharide-binding protein, ficolin-2, apolipoprotein D,kininogen-1, apolipoprotein M, thrombospondin-1, IgG Fc-binding protein,cystatin-c, alpha-1-acid glycoprotein 2, and leucine-richalpha-2-glycoprotein, and thereby, diagnosing, prognosing or monitoringor staging the progression of NAFLD.

The invention further provides a method of scoring to determine theseverity of NAFLD, the method comprising detecting and quantifying oneor more biomarkers in a biological sample obtained from the individual,wherein the one or more biomarkers is selected from apolipoprotein F,lipopolysaccharide-binding protein, ficolin-2, apolipoprotein D,kininogen-1, apolipoprotein M, thrombospondin-1, IgG Fc-binding protein,cystatin-c, alpha-1-acid glycoprotein 2, and leucine-richalpha-2-glycoprotein and wherein the level of biomarkers is compared toa control sample or reference sample/level to derive a score.

The invention additionally provides a method of treating NAFLD, themethod comprising diagnosing NAFLD in an individual, by a method asdescribed above and administering an agent or carrying out a treatmentregimen effective to treat NAFLD to the individual.

The invention will now be described in more detail, by way of exampleand not limitation, and by reference to the accompanying drawings. Manyequivalent modifications and variations will be apparent, to thoseskilled in the art when given this disclosure. Accordingly, theexemplary embodiments of the invention set forth are considered to beillustrative and not limiting. Various changes to the describedembodiments may be made without departing from the scope of theinvention as defined in the claims. All documents cited herein, whethersupra or infra, are expressly incorporated by reference in theirentirety.

The present invention includes the combination of the aspects andpreferred features described except where such a combination is clearlyimpermissible or is stated to be expressly avoided. As used in thisspecification and the appended claims, the singular forms “a”, “an”, and“the” include plural referents unless the content clearly dictatesotherwise. Thus, for example, reference to “a biomarker” includes two ormore such biomarkers.

DESCRIPTION OF THE FIGURES

FIG. 1—Magnified regions of representative 2D-PAGE gels showing thedecrease in expression of biomarkers across NAFLD stages. Three novelbiomarkers are shown as an example and the relative position of theidentified protein is circled. Stages of NAFLD from left to right aresteatosis (NAFL), NASH with no fibrosis (F0) and NASH with advancedfibrosis (F3). (a) apolipoprotein D, (b) apolipoprotein M, (c)kininogen-1.

FIG. 2—Western blot densitometry data for novel biomarkers using patientserum samples with increasing NAFLD severity. (a) apolipoprotein D; thisbiomarker is high in healthy controls and consistently lower in allstages of NAFLD suggesting that it would be useful as an early NAFLDbiomarker. (b) apolipoprotein M; the biomarker shows a slight decreasefrom steatosis (NAFL) to NASH F0 but reduces markedly in expression inNASH F3 suggesting that it is most useful at determining hepaticfibrosis. (c) kininogen-1; a consistent decrease across all NAFLDstages. (d) apolipoprotein F; a consistent decrease across all NAFLDstages.

FIG. 3—Mass spectrometric relative quantitation data from the use oftandem mass tags (TMT; 10-plex) for novel NAFLD biomarkers using serumsamples from healthy individuals (control) and patients with steatosis(NAFL) and NASH (with Kleiner-Brunt scores of F0, F1 and F3). (3 a)Apolipoprotein F (APO-F) shows a decrease in its concentration withincreasing NAFLD severity. (3 b) Adiponectin decreases in itsconcentration from control to steatosis (NAFL) to NASH but is constantin all fibrosis stages of NASH suggesting that it is a good NAFLDbiomarker. (3 c) Ficolin-2 shows some decrease in NAFLD with its lowestlevels in fibrosis stage F3. (3 d) Thrombospondin-1 increases fromcontrol to steatosis (NAFL) and across all stages of NAFLD.

FIG. 4—Parallel reaction monitoring (PRM) based mass spectrometricquantitation data for selected potential biomarkers. (4 a) Absoluteconcentration of APO-F using three different tryptic peptides in itssequence. A digest of the serum samples (100 ng) was used to quantifyAPO-F using the IGNIS LC-MS method. (4 b-i) Relative quantitation ofbiomarkers using PRM for (4 b) ficolin-2, (4 c) thrombospondin-1, (4 d)adiponectin, (4 e) IgG Fc-binding protein, (4 f) cystatin-C, (4 g)lipopolysaccharide-binding protein, (4 h) alpha-1-acid glycoprotein 2,(4 i) leucine-rich alpha-2-glycoprotein and (4 j) apolipoprotein D. Allthe samples were analyzed in two technical replicates.

DETAILED DESCRIPTION

The present inventors have identified particular biomarkers that can beused alone or in combination in the diagnosis, prognosis or monitoringor staging the progression of non-alcoholic fatty liver disease (NAFLD).These biomarkers can be used in particular in staging NAFLD, to assessan individual as having non-alcoholic fatty liver (NAFL), non-alcoholicsteatohepatitis (NASH), hepatic fibrosis and/or cirrhosis, associatedwith NAFLD. The identified biomarkers can be used in order to score theseverity of NAFLD. These markers are also useful in methods comprisingthe diagnosis, monitoring or staging of NAFLD, and subsequently treatingthe individual, to treat NAFLD.

The present invention is directed to the analysis of one or morebiomarkers in a sample taken from an individual. Typically, the sampleis a blood sample, serum sample or plasma sample, although it mayinclude cells, cell lysates, urine, amniotic fluid and other biologicalfluids. A blood sample may be capillary, venous or arterial blood or aplasma or serum sample. The sample may be a neat sample or extractedfrom a dried blood spot or other solid medium. Collection of samples maybe performed using tubes (including vacutainers) or the sample can beblotted onto a solid medium such as dried blood spots (using capillaryblood) on filter paper, cellulose membranes or the like. In the case ofdried blood spots (DBS) this can be taken from a patient by prickingtheir finger with a lancet and transferring one or more drops ofcapillary blood onto a filter paper, cellulose membrane or equivalentsample support medium which allows easier sample handling and sampletransportation as well as being more favored by the patient compared tovenipuncture. Capillary blood could also be collected in specializedtubes such as the Microtainer, Microvette, Monovette, Minivette orMultivette.

The individual is typically a human subject. The individual may displayone or more symptoms of NAFLD and/or may previously have been diagnosedwith NAFLD, where the present methods are used in monitoring or stagingof NAFLD. Additional parameters may have previously been used, or beused in combination with the methods of the present invention to aid inthe assessment of NAFLD. Such parameters include patient age, patientgender, patient weight, patient height, body mass index (BMI), evidenceof rapid weight loss/weight gain, eating habits, alcohol intake,medication records, patient exercise/activity history, ethnicity, anddisease history (including history of diabetes including type IIdiabetes, cardiovascular disease, obesity, gastric bypass surgery,polycystic ovary syndrome, sleep apnoea, hypothyroidism,hypopituitarism, and metabolic syndrome which usually includes at leastthree of the following factors: increased waist circumference,hypertriglyceridemia, hypertension, high fasting glucose, and a lowhigh-density lipoprotein (HDL) level).

In accordance with the present invention, one or more biomarkers aredetected and quantified, at least one biomarker being typically selectedfrom apolipoprotein F, lipopolysaccharide-binding protein, ficolin-2,apolipoprotein D, kininogen-1, apolipoprotein M, thrombospondin-1, IgG FC-binding protein, cystatin-C, alpha-1-acid glycoprotein 2, andleucine-rich alpha-2-glycoprotein.

The biomarkers, and their database accession number in the Uniprotdatabase are listed below. The protein sequences as disclosed herein arewith reference to the Uniprot sequence as of 31 Jul. 2016. Thus, belowis a list of our biomarkers with their accession numbers (inparentheses) and alternative names (in italics):

apolipoprotein-F (Q13790) Apo-F, Lipid transfer inhibitor protein, LTIP;

apolipoprotein-M (095445) Apo-M, ApoM, Protein G3a;

apolipoprotein-D (P05090) Apo-D, ApoD;

kininogen-1 (P01042) Alpha-2-thiol proteinase inhibitor, Fitzgeraldfactor, High molecular weight kininogen, HMWK,Williams-Fitzgerald-Flaujeac factor Kininogen-1 is Cleaved into thefollowing 6 chains: 1) Kininogen-1 heavy chain, 2) T-kinin; Alternativename(s): Ile-Ser-Bradykinin, 3) Bradykinin; Alternative name(s):Kallidin I, 4) Lysyl-bradykinin, Alternative name(s): Kallidin II, 5)Kininogen-1 light chain, 6) Low molecular weight growth-promotingfactor;

ficolin-2 (Q15485) 37 kDa elastin-binding protein, Collagen/fibrinogendomain-containing protein 2, EBP-37, Ficolin-B, Ficolin-beta, Hucolin,L-ficolin, Serum lectin p35;

thrombospondin-1 (P07996);

IgG Fc-binding protein (Q9Y6R7) Fcgamma-binding protein antigen,FcgammaBP cystatin-c (P01034) Cystatin-3, Gamma-trace, Neuroendocrinebasic polypeptide, Post-gamma-globulin;

lipopolysaccharide-binding protein (P18428) LBP;

alpha-1-acid glycoprotein 2 (P19652) AGP 2, Orosomucoid-2, OMD 2; and

leucine-rich alpha-2-glycoprotein (P02750) LRG.

In accordance with the present invention, the inventors have identifiedthat levels of certain of these proteins decrease with increasing NAFLDseverity. In particular, apolipoprotein F, alpha-1-acid glycoprotein 2,ficolin-2 and leucine-rich alpha-2-glycoprotein all demonstratedecreasing levels of protein with increasing NAFLD severity. Incontrast, levels of cystatin C, lipopolysaccharide-binding protein, IgGFc-binding protein and thrombospondin-1 show increasing levels withincreasing NAFLD severity.

In a preferred aspect of the present invention, the one or more markeris selected from apolipoprotein F, apolipoprotein D, kininogen-1,ficolin-2, apolipoprotein M and thrombospondin-1. A particularlypreferred biomarker is apolipoprotein F.

Apolipoprotein F shows a decrease in concentration with increasing NAFLDseverity. Accordingly, monitoring changes in the level of this proteinin an individual provides an indication of the progress of NAFLD throughthe stages.

Apolipoprotein D demonstrates high levels in healthy controls, andconsistency lower levels in all stages of NAFLD. Accordingly, thismarker can be used as an early NAFLD biomarker. A decreasing level ofapolipoprotein D, as compared with a control sample or referencesample/level, indicates increasing severity of NAFLD. Apolipoprotein Mshows a slight decrease from steatosis (NAFL) to NASH F0, but reduces anexpression in NASH F3. Accordingly, apolipoprotein M is useful indetermining progress to hepatic fibrosis. Kininogen-1 shows a consistentdecrease across all NAFLD stages.

Ficolin-2 shows a decrease in NAFLD stages, showing its lowest levels infibrosis stage F3.

Thrombospondin-1 increases from control levels to steatosis (NAFL) andacross all stages of NAFLD.

In a preferred aspect of the present invention, the at least onebiomarker comprises apolipoprotein F, lipopolysaccharide-bindingprotein, and/or ficolin-2. In an additional preferred aspect of theinvention, the at least one biomarker comprises apolipoprotein F,lipopolysaccharide-binding protein, ficolin-2, and/or apolipoprotein D.

In another preferred aspect of the present invention, the presentinvention provides for analysis of at least two, at least three, atleast four or at least five or more biomarkers and thereby, diagnosing,prognosing or monitoring or staging the progression of NAFLD. Thus, thebiomarkers identified above are preferably used in combination with oneor more additional biomarkers.

For example, a combination of markers can be used to provide moreprecise staging of NAFLD. Such biomarkers may be selected fromapolipoprotein F, lipopolysaccharide-binding protein, ficolin-2,apolipoprotein D, kininogen-1, apolipoprotein M, thrombospondin-1, IgGFc-binding protein, cystatin-c, alpha-1-acid glycoprotein 2, andleucine-rich alpha-2-glycoprotein. A combination of markers used inaccordance with the invention preferably comprises the biomarkerapolipoprotein F and one or more additional biomarkers selected fromlipopolysaccharide-binding protein, ficolin-2, apolipoprotein D,kininogen-1, apolipoprotein M, thrombospondin-1, IgG Fc-binding protein,cystatin-c, alpha-1-acid glycoprotein 2, and leucine-richalpha-2-glycoprotein. A preferred combination comprises apolipoprotein Fand one or more, such as two, three or four additional biomarkersselected from lipopolysaccharide-binding protein, ficolin-2,apolipoprotein D, alpha-1-acid glycoprotein 2 and thrombospondin-1.

Additional combinations of markers that may be used in accordance withthe invention include (i) one or more biomarkers selected fromapolipoprotein F, lipopolysaccharide-binding protein, and ficolin-2, incombination with one or more biomarkers selected from apolipoprotein D,alpha-1-acid glycoprotein 2 and thrombospondin-1; and (ii) one or morebiomarkers selected from apolipoprotein F, lipopolysaccharide-bindingprotein, and ficolin-2, in combination with one or more biomarkersselected from apolipoprotein D, alpha-1-acid glycoprotein 2 andthrombospondin-1. Alternatively, additional biomarkers may be combinedwith the one or more biomarkers of the invention.

In a preferred aspect of the present invention, the method comprisesanalysis of at least apolipoprotein F and lipopolysaccharide-bindingprotein; apolipoprotein F and ficolin-2; apolipoprotein F andapolipoprotein Dlipopolysaccharide-binding protein and ficolin-2;lipopolysaccharide-binding protein and apolipoprotein D; apolipoproteinD and ficolin-2; apolipoprotein F, lipopolysaccharide-binding proteinand ficolin-2; or apolipoprotein F, apolipoprotein D,lipopolysaccharide-binding protein and ficolin-2

The present methods may be used in the diagnosis of NAFLD, or in theprognosis of NAFLD, for example, to establish the severity of disease.In a preferred aspect of the present invention, the methods can be usedin the staging of NAFLD, and in particular, in the staging of theindividual as suffering from non-alcoholic fatty liver (NAFL), alsoreferred to as steatosis. A preferred biomarker for early staging ofNAFLD and staging as NAFL is apolipoprotein D. The methods may also beused to stage NAFLD as non-alcoholic steatohepatitis (NASH). A preferredbiomarker for staging severity of NAFLD and staging as NASH isapolipoprotein M. The methods may also be used to stage the individualas suffering from hepatic fibrosis, including the severity of fibrosis.In a further aspect of the present invention, in addition to theanalysis of the one or more biomarkers as disclosed above, the inventionalso provides the analysis of one or more further biomarkers. The one ormore further biomarkers may be selected from the further biomarkersidentified according to the invention as described below.

The one or more further biomarkers may comprise keratin type Icytoskeletal 18, accession number in parentheses; alternative names initalics: (P05783) Cell proliferation-inducing gene 46 protein,Cytokeratin-18, CK-18, Keratin-18, K18 and/or adiponectin (Q15848) 30kDa adipocyte complement-related protein, Adipocyte complement-related30 kDa protein, ACRP30, Adipocyte, C1q and collagen domain-containingprotein, Adipose most abundant gene transcript 1 protein, apM-1,Gelatin-binding protein.

Adiponectin is a particularly preferred further biomarker. The resultsdescribed herein show that adiponectin decreases in concentration fromcontrol to steatosis (NAFL) to NASH, but is constant in the fibrosisstages of NASH. Accordingly, this is a useful early marker for NAFL andprogression to NASH. A decreasing level of adiponectin, as compared witha control sample or reference sample/level, thus indicates increasingseverity of NAFLD as it progresses up to the non-alcoholicsteatohepatitis stage.

Accordingly, a method of the present invention may comprise analysis ofat least apolipoprotein F and adiponectin; lipopolysaccharide-bindingprotein and adiponectin; ficolin-2 and adiponectin; apolipoprotein D andadiponectin; apolipoprotein F, apolipoprotein D and adiponectin;apolipoprotein F, lipopolysaccharide-binding protein and adiponectin;apolipoprotein F, ficolin-2 and adiponectin; lipopolysaccharide-bindingprotein, ficolin-2 and adiponectin; apolipoprotein D,lipopolysaccharide-binding protein and adiponectin; apolipoprotein Dficolin-2 and adiponectin; or apolipoprotein F,lipopolysaccharide-binding protein, ficolin-2 and adiponectin.

The invention also provides for the assessment of cleavage products ofone or more proteins. Suitable proteins for analysis of cleavageproducts include kininogen-1, keratin type I cytoskeletal 18, andangiotensinogen. Typically, the amount of cleavage products for thesepeptides increases with severity of NAFLD, such that monitoring oflevels of the cleavage products can be used as part of the methods ofthe present invention.

The present methods can also be used to monitor the progress of NAFLD,for example, in a patient diagnosed with NAFLD. The method can berepeated at suitable intervals, such as once a month, once every twomonths, once every three months, once every six months, once a year,once every two years or once every three years in order to monitor theprogress of NAFLD, and in particular, to monitor changes in thebiomarker(s), associated with progression to a different stage of NAFLD.Such monitoring can also be used in assessment of the effectiveness oftherapy for such patients.

The one or more biomarkers of the present invention can be assessed byany suitable technique, including for example immunoassays. In aparticularly preferred aspect of the present invention, the methods usemass spectrometry to assess the one or more biomarkers.

In specific embodiments, the one or more biomarkers are quantified usingantibody-free approaches such as, but not limited to, Parallel ReactionMonitoring (PRM), Selected Reaction Monitoring (SRM), Multiple ReactionMonitoring (MRM) or multi-stage fragmentation in MRM-cubed (MRM³) usingmass spectrometry.

The biological sample (either capillary/venous/arterial blood, serum orplasma) is digested with a protease such as, but not limited to,trypsin, chymotrypsin, Arg-C, Asp-N, clostripain, elastase, Glu-C,Lys-C, Lys-N, pepsin, protein endopeptidase and Staphylococcus protease,but in specific embodiments the enzyme is trypsin. In the case oftrypsin, this also includes immobilized trypsin for higher throughputdigestion such as Flash digest from Perfinity Biosciences (also calledSMART Digest from Thermo Scientific). The cleavage properties of theseenzymes are known (for example, trypsin cleaves after lysine, K orarginine, R) and so the resulting peptides are determined manually orusing a software (such as Skyline, PinPoint etc) to digest the proteinsin-silico. Typically peptides shorter than 7 amino acids are excludedsince they are unlikely to be unique. Typically peptides longer than 25amino acids are excluded since synthetic peptides as standards arelikely to be expensive. Although peptides between 7 to 25 amino acidsare typically selected for analysis, peptides shorter than 7 amino acidsand longer than 25 amino acids should not be entirely excluded. Whenusing trypsin, typically peptides with no missed cleavages are selectedbut peptides with one or more missed cleavages can be considered for anyenzyme. Typically the peptides are selected which are not modified (e.gglycosylated, phosphorylated, etc) and methionine, M, is avoided sincethis amino acid may or may not be oxidized and these modifications wouldchange the mass of the peptide. Modified peptides should not be entirelyexcluded if the mass of the modification (e.g. glycosylation,phosphorylation, oxidation, carbamidomethylation etc) is known.

Online databases such as, but not limited to, PeptideAtlas and theGlobal Proteome Machine database (gpmdb), help to determine the commonlyobserved peptides for the proteins of interest. MRMaid can be used tohelp design assays for mass spectrometry based targeted quantitation bysuggesting peptides and MS² ions to monitor based on experimentalspectra from the PRIDE database. Protein BLAST is used to check theuniqueness of the selected peptide. If a peptide is not unique, thenanother peptide within the biomarker sequence would be selected.

Prior to mass spectrometry, digested peptides can be fractionated usingphosphopeptide enrichment, glycopeptide enrichment, high pH reversedphase fractionation or the like. Peptides can be separated by reversedphase liquid chromatography, hydrophilic interaction liquidchromatography (HILIC), ion-exchange chromatography, isoelectricfocusing or the like but in specific embodiments separation is byreversed phase liquid chromatography using a carbon-18 (C-18) column. Inthe case of very low abundant biomarkers, samples can be fractionatedusing pH based reversed-phase fractionation, biomarkers could beenriched using immunoprecipitation or high abundant proteins could bedepleted (using immunoprecipitation or dye-affinity methods such asCibacron-blue for the depletion of albumin in serum and/or plasmasamples).

SRM/MRM is performed using a triple quadrupole mass spectrometer. Thefirst quadrupole filters the preselected peptide (MS¹ ion) of theprotein of interest, the second quadrupole fragments this peptide andthe third quadrupole filters a fragment or fragments (MS² ions) of thepeptide for detection. PRM is performed using a hybridquadrupole-Orbitrap mass spectrometer such as a Thermo Q Exactive andits variants or a Thermo Fusion and its variants. In PRM, the quadrupolefilters the preselected peptide (MS¹ ion) of the protein of interest,the higher-energy collisional dissociation (HCD) cell fragments thispeptide and the Orbitrap is able to detect all fragments (MS² ions) ofthe peptide due to its high resolution. MRM³ is performed using a QTRAPmass spectrometer. In MRM³, the first quadrupole filters the preselectedpeptide (MS² ion) of the protein of interest, the second quadrupolefragments this peptide, then the fragment ions (MS² ions) are trappedafter being isolated in the linear ion trap followed by excitation toperform a second fragmentation step with MS³ ions to give a higher levelof sensitivity. In specific embodiments, the technique includes anytandem mass spectrometer capable of MS/MS with sample ionization carriedout using electrospray ionization (ESI) but may also be performed usingother ionization methods.

The area under the curve (AUC) of the fragment ions can be used torelatively quantify the levels of the proteins. Known amounts ofsynthetic pure heavy-labelled peptides with the same sequence as thepreselected peptides can be used to establish a calibration curve tohelp determine the absolute concentration of the proteins. In moredetail, detection of these peptides can be carried out using SRM, MRM,MRM³, PRM or the like where a mass spectrometer filters for thepreselected peptide (MS¹ ion), fragments the peptide and the peptidefragments (MS² ions) are detected. The AUC for these MS' ions are usedto quantify the biomarkers. For absolute quantitation, known amounts ofsynthetic peptides are used which can be isotopically labelled.Synthesis is typically performed from the C-terminus to the N-terminusof the peptide. The synthesis cycle comprises: a) Loading of theC-terminal amino acid to the solid support phase (polystyrene resin).This amino acid is protected at the N-terminus to avoid unwantedreactions; b) Removal of N-terminal protection group (PG) of this aminoacid; c) Activation of C-terminus of the next amino acid (N-terminuscarries protection group); d) Coupling reaction of these two aminoacids; e) Re-start of cycle at b) or cleavage of full-length peptidefrom resin. Peptides can be crude or having a purity of at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% or atleast 98%. Peptides can be synthetic peptides, IGNIS peptides, PEPotecpeptides, AQUA peptides, SpikeTide peptides, Protein Epitope SignatureTag (QPrEST) peptides, microwave-assisted solid phase synthesizedpeptides, concatenated signature peptides encoded by QconCAT genes orthe like. Both detection and/or enrichment of the endogenous peptides inthe biological sample can be achieved using anti-peptide antibodies,Stable Isotope Standard Capture with Anti-Peptide Antibodies (SISCAPA)or the like where the anti-peptide antibody is raised against thepreselcted peptide. In addition to isotopically labelled peptides,quantitation of biomarkers can also be achieved by label-free massspectrometry based quantitation (using the Progenesis LC-MS software,Skyline software or the like) or by labelling the proteins in thebiological samples using isotope tags such as tandem mass tags (TMT),isobaric tags for relative and absolute quantitation (iTRAQ),isotope-coded affinity tags (ICAT) or the like.

Absolute quantitation can be carried out using the HeavyPeptide IGNISPrime Custom Peptide Quantitation Kit from Life Technologies with thedigestion of IGNIS Prime peptides using either trypsin or immobilizedtrypsin such as Flash digest from Perfinity Biosciences (also calledSMART Digest from Thermo Scientific).

Using apolipoprotein F (APO-F) as an example the workflow for peptideselection is described and this can be followed for any of thebiomarkers listed. The unprocessed precursor of APO-F has 326 aminoacids. Amino acids 1-35 are for a signal peptide, amino acids 36-164 arefor a propeptide and amino acids are for the functional APO-F. In-silicodigestion of the functional APO-F sequence shows four peptides which arebetween the criteria of 7 to 25 amino acids: SLPTEDCENEK (SEQ ID NO: 1)(m/z 661.2825, 2+ or m/z 441.1907, 3+), SGVQQLIQYYQDQK (SEQ ID NO: 2)(m/z 849.4283, 2+ or m/z 566.6213, 3+), DANISQPETTK (SEQ ID NO: 3) (m/z602.2962, 2+, m/z 401.8666, 3+), SYDLDPGAGSLEI (SEQ ID NO: 4) (m/z668.8170, 2+, m/z 446.2138, 3+). All these peptides can be targeted bymass spectrometry using SRM, MRM, PRM, MRM³ or the like by targeting theMS¹ ions shown above in parentheses. In the case of DANISQPETTK (SEQ IDNO: 3), the second threonine, T, in its sequence is O-glycosylated andso would not normally be chosen as a suitable peptide. However,DANISQPETTK (SEQ ID NO: 3) should not be ruled out if the protein isdeglycosylated.

In addition to the detection of the biomarkers themselves, in someembodiments, the methods of the invention are directed to the detectionof fragments of the particular biomarkers. For example, some proteinssuch as keratin type I skeletal 18 and kininogen-1 are cleaved, with thelevel of cleavage increasing in NAFLD. Accordingly, for these proteins,monitoring for the cleavage products provides a marker for NAFLD and/orthe stage of disease. In preferred embodiments, cleavage products aredetected by mass spectrometry methods. When detecting cleavage products,the initial protease digestion step may not be conducted.

In the case of keratin type I cytoskeletal 18, this protein is cleavedby a caspase between amino acids 397/398 of the complete unprocessedsequence (i.e. between amino acids 396/397 of the functional keratintype I cytoskeletal 18 after the initiator methionine is removed) andthis cleavage increases with NAFLD. The tryptic peptideLLEDGEDFNLGDALDSSNSMQTIQK (SEQ ID NO: 5) covers this cleavage region andcan be targeted by mass spectrometry using SRM, MRM, PRM, MRM³ or thelike. The three serine (S) amino acids in the sequence are known to bephosphorylated and methionine (M) could be oxidized so the m/z of thispeptide should be calculated if these four amino acids are both modifiedand unmodified. Levels of this peptide decrease with increasing NAFLDseverity. In addition the cleaved tryptic peptide LLEDGEDFNLGDALD (SEQID NO: 6) can be targeted by mass spectrometry using SRM, MRM, PRM, MRM³or the like by targeting a peptide of m/z 818.3729, 2+ or m/z 545.9177,3+. Levels of this peptide increase with increasing NAFLD severity. Thecleaved tryptic peptide SSNSMQTIQK (SEQ ID NO: 7) can also be targetedby mass spectrometry using SRM, MRM, PRM, MRM³ or the like and alsoincrease with increasing NAFLD severity although as before the m/z ofthis peptide should be calculated if the serine (S) and methionine (M)amino acids are both modified and unmodified.

Kininogen-1 was found to be a novel biomarker for NAFLD. Kininogen-1cleaves into the following 6 chains all of which may be potentialbiomarkers for NAFLD: kininogen-1 heavy chain, T-kinin, bradykinin,lysyl-bradykinin, kininogen-1 light chain and low molecular weightgrowth-promoting factor. T-kinin (sequence ISLMKRPPGFSPFR (SEQ ID NO:8); m/z 816.9558, 2+; m/z 544.9729, 3+), bradykinin (sequence RPPGFSPFR(SEQ ID NO: 9); m/z 530.7880, 2+; m/z 354.1944, 3+) and lysyl-bradykinin(sequence KRPPGFSPFR (SEQ ID NO: 10); m/z 594.8355, 2+; m/z 396.8927,3+) are short peptides which can be targeted by mass spectrometry usingeither SRM, MRM, PRM, MRM³ or the like. These sequences are targetedwithout predigesting the sample with trypsin or other enzymes.Bradykinin is released from kininogen by plasma kallikrein.Hydroxylation of the second proline (P) in the sequences above(RPP[+16]GFSPFR (SEQ ID NO: 9); m/z 538.7854, 2+; m/z 359.5236, 3+)occurs prior to the release of bradykinin which would additionally needto be considered for MS¹ ion selection.

In a similar way to keratin type I cytoskeletal 18 and kininogen-1,peptides for other proteins which cleave with fatty liver and/orfibrosis can be selected which cover the cleavage regions and are oneither side of the cleavage region. For example, alpha 2 macroglobulinand cleaved/intact complement C3 are proteins with a fibrosis-dependentthioester cleavage region.

Hemoglobin A1c (HbA1c, glycated hemoglobin) is a component of hemoglobinwhich binds glucose and elevated levels indicate poor glucose controlover 6-8 weeks in diabetics. Type II diabetes is a risk factor in NAFLDand so HbA1c levels can also be measured in addition to the biomarkersdisclosed herein. In HbA1c, glucose is attached to the N terminal valine(V) of the hemoglobin beta chain. Unprocessed hemoglobin beta chain is147 amino acids in length and the first amino acid is an initiatormethionine which is removed. The remaining amino acids from 2-147 makeup the functional hemoglobin beta chain. The valine (V) amino acid inposition 2 is the glycation site for HbA1c. HbA1c is usually measured inthe clinic by HPLC but could also be analyzed by targeted massspectrometry using SRM, MRM, PRM, MRM³ or the like. If the proteins in abiological sample from a NAFLD or diabetic patient are digested withtrypsin, the N terminal peptide from hemoglobin beta chain has thesequence VHLTPEEK (SEQ ID NO: 11) (m/z 476.7585, 2+; m/z 318.1748, 3+)which can be targeted by mass spectrometry. The presence of this peptideat m/z 476.7585 (2+) or m/z 318.1748 (3+) would indicate hemoglobin betachain which is not glycated at the N terminal valine (V) whereas theabsence of this peptide would suggest glycation of hemoglobin beta chainat the N terminal valine (V). The glycated peptide will have additionalmass of glucose (with loss of one water molecule) with the sequenceV[+162.1]HLTPEEK (SEQ ID NO: 11) (m/z 557.7850, 2+; m/z 372.1924, 3+).This can be used to assess glucose control in a patient and formonitoring type II diabetes which is a major risk factor for NAFLD. Thisapproach can also be used to check other glycation sites in hemoglobinalpha and beta chains as well as other proteins.

Angiotensinogen is also a biomarker of fibrosis. The following peptidehormones are derived from angiotensinogen: angiotensin-1 (DRVYIHPFHL(SEQ ID NO: 12); m/z 648.8460, 2+; m/z 432.8998, 3+), angiotensin 1-9(DRVYIHPFH (SEQ ID NO: 13); m/z 592.3040, 2+; m/z 395.2051, 3+),angiotensin-2 (DRVYIHPF (SEQ ID NO: 14); m/z 523.7745, 2+; m/z 349.5188,3+), angiotensin 1-7 (DRVYIHP (SEQ ID NO: 15); m/z 450.2403, 2+; m/z300.4960, 3+), angiotensin 1-5 (DRVYI (SEQ ID NO: 16); m/z 333.1845, 2+;m/z 222.4588, 3+; not unique and too short), angiotensin 1-4 (DRVY (SEQID NO: 17); m/z 276.6425, 2+; m/z 184.7641, 3+; not unique and tooshort), angiotensin-3 (RVYIHPF (SEQ ID NO: 18); m/z 466.2611, 2+; m/z311.1765, 3+) and angiotensin-4 (VYIHPF (SEQ ID NO: 19); m/z 388.2105,2+; m/z 259.1428, 3+). Angiotensin-2 is a known hepatic stellate cellactivator in liver fibrosis. These peptides can be targeted by massspectrometry using either SRM, MRM, PRM, MRM³ or the like. Thesesequences must be targeted without predigesting the sample with trypsinor other enzymes.

Since the peptides from kininogen-1 and angiotensinogen are liberatednaturally from their processing, they can be detected without the needfor cleavage with enzyme (e.g. trypsin, chymotrypsin etc). The peptidescan be detected from the biological sample by removal of proteins with ahigher molecular weight using chromatography, molecular weight cut offfilters, size exclusion or the like and then targeting the peptide bymass spectrometry.

The detection methods of the invention may also comprise use of an agentwherein the agent specifically detects proteins or peptides of interest.The agent could be an antibody or functional equivalent thereof thatbinds proteins or peptides under analysis (i.e. anti-peptide antibody).These antibodies may be used to perform an immunoassay such as, but notlimited to, enzyme linked immunosorbent assay (ELISA),radio-immunoassay, protein dot blot, Western blot, turbidimetry,nephelometry and the like. The kit may further comprise at least onetarget specifically for detecting another gene or gene product useful asa prognostic indicator.

The biomarkers may also be detected by suitable immunoassay using agentsthat specifically bind the marker of interest. Such binding agentsinclude immunoglobulins and functional equivalents of immunoglobulinsthat specifically bind to the biomarkers of interest. The terms“immunoglobulin” and “antibody” are used interchangeably and in theirbroadest sense herein. Thus, they encompass intact monoclonalantibodies, polyclonal antibodies, antibody phage display (APD),multispecific antibodies (e.g., bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments, so long as theyexhibit the desired biological activity. APD involves bacteriophagegenetic engineering and multiple rounds of phage propagation and antigenguided selection.

In addition to using antibodies to detect the biomarkers, antibodies canalso be used to enrich the biomarkers in the biological sample to helpwith other detection/quantitation methods. This includes techniques suchas immunoprecipitation using Protein A, Protein G, Protein A/G orstreptavidin/avidin beads for biotinylated antibodies. Usingimmunoprecipitation antibodies against the target biomarkers couldoptionally be crosslinked onto the beads using a crosslinker such asdisuccinmidyl suberate (DSS) to avoid the presence of the antibody inthe elution. Alternatively Mass Spectrometric Immunoassay (MSIA) can beused for downstream mass spectrometry analysis of NAFLD-ASSOCIATEDbiomarkers using SRM, MRM, MRM³, PRM or the like. MSIA tips could haveeither Protein A, Protein G or Protein A/G for the capture of antibodiesto the target biomarker. MSIA tips could have streptavidin or avidin forthe capture of biotinylated antibodies to the target biomarker. MSIA isparticular useful for very low abundant biomarkers which cannot beusually detected without enrichment (such as keratin type I cytoskeletal18).

The invention further provides methods for detecting the presence ofand/or measuring a level of one or more biomarkers of interest in abiological sample, using an antibody specific for the biomarker ofinterest. Specifically, the method for detecting the presence of thebiomarker of interest in a biological sample may comprise the step ofcontacting the sample with a monoclonal antibody and detecting thebinding of the antibody with the biomarker in the sample. Morespecifically, the antibody may be labeled so as to produce a detectablesignal using compounds including, but not limited to, a radiolabel, anenzyme, a chromophore and a fluorophore.

Detection of specific binding of an antibody specific for the protein ofinterest, or a functional equivalent thereof, when compared to asuitable control, is an indication that the biomarker is present in thesample. Suitable controls include a sample known not to contain theproteins of interest and a sample contacted with an antibody notspecific for the encoded protein, e.g., an anti-idiotype antibody. Avariety of methods to detect specific antibody-antigen interactions areknown in the art and may be used in the method, including, but notlimited to, standard immunohistological methods, immunoprecipitation, anenzyme immunoassay, and a radioimmunoassay. In general, the specificantibody will be detectably labeled, either directly or indirectly.Direct labels include radioisotopes; enzymes whose products aredetectable (e.g., luciferase, 3-galactosidase, and the like);fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine,phycoerythrin, and the like); fluorescence emitting metals (e.g., 112Eu,or others of the lanthanide series, attached to the antibody throughmetal chelating groups such as EDTA); chemiluminescent compounds (e.g.,luminol, isoluminol, acridinium salts, and the like); bioluminescentcompounds (e.g., luciferin, aequorin (green fluorescent protein), andthe like). The antibody may be attached (coupled) to an insolublesupport, such as a polystyrene plate or a bead. Indirect labels includesecond antibodies specific for antibodies specific for the encodedprotein (“first specific antibody”), wherein the second antibody islabeled as described above; and members of specific binding pairs, e.g.,biotin-avidin, and the like. The biological sample may be brought intocontact with and immobilized on a solid support or carrier, such asfilter paper, nitrocellulose membrane or cellulose membrane, that iscapable of immobilizing cells, cell particles, or soluble proteins. Thesupport may then be washed with suitable buffers or solvents (such asacetone to dissolve the cellulose membrane and precipitate the proteinsin the biological sample), followed by digesting the proteins in thesample to peptides using enzymes and then detecting and quantifying thebiomarkers by mass spectrometry or contacting with a detectably-labeledfirst specific antibody. Detection methods are known in the art and willbe chosen as appropriate to the signal emitted by the detectable label.Detection is generally accomplished in comparison to suitable controlsand to appropriate standards.

The detection methods of the invention may also comprise use of an agentto detect changes in post-translational modifications of the biomarkersin NAFLD and across NAFLD stages. These post-translational modificationsmay include N-glycosylation, 0-glycosylation and phosphorylation.

In another embodiment, the current invention provides a method ofdetermining the prognosis of NAFLD/fibrosis, comprising: (a) determiningthe level of a protein selected from apolipoprotein F,lipopolysaccharide-binding protein, ficolin-2, apolipoprotein D,kininogen-1, apolipoprotein M, thrombospondin-1, IgG Fc-binding protein,cystatin-c, alpha-1-acid glycoprotein 2, and leucine-richalpha-2-glycoprotein in a biological sample obtained from a patient; and(b) comparing said level of (a) to a control level of said protein inorder to determine a positive or negative diagnosis of said fatty liveror fibrosis.

Methods of Therapy

In some cases in accordance with the invention, a method of treatingNAFLD is described. The method comprises diagnosing NAFLD in anindividual, detecting and quantifying one or more biomarkers in abiological sample obtained from the individual, wherein the one or morebiomarkers is selected from apolipoprotein F, lipopolysaccharide-bindingprotein, ficolin-2, apolipoprotein D, kininogen-1, apolipoprotein M,thrombospondin-1, IgG Fc-binding protein, cystatin-c, alpha-1-acidglycoprotein 2, and leucine-rich alpha-2-glycoproteinadministering anagent to the individual or carrying out a treatment regimen on theindividual and thereby treating NAFLD.

The individual typically has NAFLD, i.e. has been diagnosed as havingNAFLD, or is suspected as having NAFLD, i.e. shows the symptoms ofNAFLD. As used herein, the term “treating” includes any of following:the prevention of the disease or of one or more symptoms associated withthe disease; a reduction or prevention of the development or progressionof the disease or symptoms; and the reduction or elimination of anexisting disease or symptoms. Treatments of NAFLD/NASH are aimed atreducing liver-related and all-cause (specifically cardiovascular)morbidity and mortality. Selection of a treatment approach requiresassessment of liver disease severity. Treatment strategies that may beused according to the invention include (i) lifestyle intervention orother weight loss regime, aiming for weight loss of >7; and (ii)treatments optimising cardiovascular risk factors, including glucosecontrol in diabetics, blood pressure control, or lipid-lowering therapy.

Agents that may be administered for treatment include angiotensinconverting enzyme inhibitors (ACE-I)/angiotensin receptor blockers(ARBs), which have potential antifibrotic effects, and metformin whichmay reduce risk of liver cancer. Liraglutide is a further agent that maybe used for treatment, which appears to improve NASH beyond its effecton weight loss (LEAN study, Armstrong et al. Lancet 2015). Liverspecific therapeutic agents (such as those in phase 3 clinical trials)aimed at those patients with NASH and more advanced fibrosis (F2-3) mayalso be used. Examples include Obeticholic acid, a synthetic bile acidderivative (Intercept pharmaceuticals), Elafibranor, a PPAR a/d agonist(Genfit). There are currently a number of other drug candidates atvarious stages of development which may be used. A further treatmentstrategy which may be used in an individual diagnosed with NAFLD issurgical intervention. For example, bariatric surgery (sleevegastrectomy or roux-en-y gastric bypass) in morbidly obese patients.

Specific routes, dosages and methods of administration of thetherapeutic agents described herein may be routinely determined by themedical practitioner. These are discussed in more detail below.

Agent

The agents for use in the methods of treatment described herein may beformulated in pharmaceutical compositions. These compositions maycomprise, in addition to the therapeutically active ingredient(s), apharmaceutically acceptable excipient, carrier, diluent, buffer,stabiliser or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The pharmaceutical carrier or diluentmay be, for example, an isotonic solution.

Kits

The invention further provides a kit that comprises means (e.g.reagents) for detecting and quantifying one or more biomarkers describedherein in a biological sample from an individual and instructions foruse of the kit in accordance with methods of the invention. The kit mayalso comprise details regarding which individuals the method may becarried out upon. The kit typically contains one or more agents, (e.g.an antibody) that specifically bind to the relevant biomarker(s). Thekit may additionally comprise means for the measurement of otherlaboratory or clinical parameters. Procedures using these kits may beperformed by clinical laboratories, experimental laboratories, medicalpractitioners, or private individuals.

The kit may additionally comprise one or more other reagents orinstruments which enable the method to be carried out. Such reagents orinstruments may include one or more of the following: suitable buffer(s)(aqueous solutions), isotopically-labelled or unlabeled peptides,peptide calibration curve standards, developing reagents, enzymes,labels, reacting surfaces, means for detection, control samples,standards, instructions, interpretive information, means to isolate arelevant biomarker from a sample, means to obtain a sample from theindividual (such as a vessel or an instrument comprising a needle) or asupport comprising wells on which quantitative reactions can be done.

Further Biomarkers Identified According to the Invention

As described in the Examples, in addition to the preferred biomarkersdescribed above initially identified by 2D-PAGE and mass spectrometryand then characterised in more detail for expression in NAFLD(apolipoprotein F, kininogen-1, apolipoprotein D, apolipoprotein M,ficolin-2, alpha-1-acid glycoprotein 2, leucine-richalpha-2-glycoprotein, lipopolysaccharide-binding protein,thrombospondin-1, IgG Fc-binding protein and cystatin-c), the inventionalso provides further biomarkers for NAFLD initially identified by2D-PAGE and mass spectrometry. These further biomarkers are listedbelow.

The invention thus also provides a method of diagnosing, prognosing ormonitoring or staging the progression of non-alcoholic fatty liverdisease (NAFLD) in an individual based on detecting and quantitating oneor more of the below further biomarkers in a biological sample obtainedfrom the individual, thereby, diagnosing, prognosing or monitoring orstaging the progression of NAFLD. The invention additionally provides amethod of diagnosing, prognosing or monitoring or staging theprogression of non-alcoholic fatty liver disease (NAFLD) in anindividual, the method comprising detecting and quantifying one or morebiomarkers in a biological sample obtained from the individual, whereinthe one or more biomarkers is selected from apolipoprotein F,lipopolysaccharide-binding protein, ficolin-2, apolipoprotein D,kininogen-1, apolipoprotein M, thrombospondin-1, IgG Fc-binding protein,cystatin-c, alpha-1-acid glycoprotein 2, and leucine-richalpha-2-glycoprotein, and additionally detecting and quantifying one ormore of the below further markers, thereby, diagnosing, prognosing ormonitoring or staging the progression of NAFLD.

Using 2D-PAGE the following further biomarkers for NAFLD were identifiedin addition to preferred biomarkers apolipoprotein F, kininogen-1,apolipoprotein D and apolipoprotein M: Proteins identified in featuresincreasing with NAFLD severity included antithrombin-III,alpha-1-antitrypsin, Ig gamma-1 chain C region, complement factor B,alpha-2-macroglobulin, complement factor H, complement C1r subcomponent,complement C4-A, inter-alpha-trypsin inhibitor heavy chain H4,apolipoprotein A-IV and serum albumin. Proteins identified in featuresdecreasing with NAFLD severity included apolipoprotein C2,apolipoprotein C4, apolipoprotein M, apolipoprotein apolipoprotein A-IV,clusterin, protein APOC4-APOC2, haptoglobin, serum albumin,zinc-alpha-2-glycoprotein, thrombin light chain, transthyretin,glutathione peroxidase 3, alpha-1-antitrypsin, alpha-1-antichymotrypsin,LMW kininogen-1, complement C3, complement C4-A, C4b-binding proteinalpha chain, complement factor I light chain, complement C1rsubcomponent, Ig alpha-1 chain C region, Ig kappa chain C region, Iggamma-1 chain C region, Ig gamma-2 chain C region, inter-alpha-trypsininhibitor heavy chain H3, prothrombin, putative elongation factor1-alpha-like 3, vitamin D-binding protein, beta-2-glycoprotein 1 andalpha-2-HS-glycoprotein.

Three protein features were found to increase in intensity from NAFL toNASH F0/F1 and then decrease in intensity in NASH F3 and the proteinsidentified in these features included complement C1s subcomponent,complement C3, haptoglobin, apolipoprotein A-IV, serum albumin, Ig kappachain C region and apolipoprotein A-I. Some proteins were identified infeatures that were both increasing and decreasing in NAFLD which couldbe due to post-translational modification or differential expression ofdifferent fragments of the protein and these proteins includedalpha-1-antitrypsin, Ig gamma-1 chain C region, complement C1rsubcomponent, complement C4-A, apolipoprotein A-IV and serum albumin.

Using mass spectrometry and TMT the following further biomarkers forNAFLD were identified in addition to preferred biomarkers ficolin-2,alpha-1-acid glycoprotein 2, leucine-rich alpha-2-glycoprotein,lipopolysaccharide-binding protein, thrombospondin-1, IgG Fc-bindingprotein and cystatin-c: Proteins identified to be increasing with NAFLDseverity included Cystatin-C (P01034),Phosphatidylinositol-glycan-specific phospholipase D (P80108),GTPase-activating Rap/Ran-GAP domain-like protein 3 (Q5VVW2), Ig kappachain V-I region DEE (P01597), Ficolin-3 (075636),Alpha-2-HS-glycoprotein (P02765), Hepatocyte growth factor-like protein(P26927), Carbonic anhydrase 1 (P00915), Ig heavy chain V-II region WAH(P01824), Ig kappa chain V-I region Mev (P01612), Properdin (P27918), Igkappa chain V-III region HAH (P18135), Hemoglobin subunit beta (P68871),von Willebrand factor (P04275), Hemoglobin subunit alpha (P69905), Igkappa chain V-I region Ni (P01613), Ig kappa chain V-I region EU(P01598), Ig alpha-1 chain C region (P01876), SPARC (P09486),Fibronectin (P02751), Platelet basic protein (P02775) and Plateletfactor 4 (P02776). Proteins identified to be decreasing with NAFLDseverity included Pregnancy zone protein (P20742), Apolipoprotein (a)(P08519), Coagulation factor XIII A chain (P00488), Galectin-3-bindingprotein (Q08380), Adiponectin (Q15848), Actin cytoplasmic 1 (P60709),Apolipoprotein A-II (P02652), Alpha-2-antiplasmin (P08697), Peptidaseinhibitor 16 (Q6UXB8), Zinc-alpha-2-glycoprotein (P25311), Alpha-1-acidglycoprotein 2 (P19652), Coagulation factor XI (P03951), Serum amyloidP-component (P02743), Protein Z-dependent protease inhibitor (Q9UK55),Monocyte differentiation antigen CD14 (P08571), Insulin-like growthfactor-binding protein complex acid labile subunit (P35858) andApolipoprotein L1 (014791).

One or more of the following biomarkers may also be analysed in additionto the biomarkers/biomarker combinations of the invention in the methodsprovided herein: afamin, alanine aminotransferase (ALT),alpha-1B-glycoprotein, alpha-1-microglobulin, alphafetoprotein (AFP),angiotensin 2, angiotensinogen, apolipoprotein A-1, apolipoprotein C1,apolipoprotein C2, apolipoprotein E, asialoglycoprotein receptor 1,asialoglycoprotein receptor 2, aspartate aminotransferase (AST), beta 2microglobulin, bilirubin, biotinidase, blood cholesterol, blood glucose(fasting and/or postprandial), blood triglycerides, blood urea nitrogen(BUN), bradykinin, C-reactive protein (CRP), carboxypeptidase N2,C4b-binding protein beta chain, CD5 antigen like protein (CDSL),ceruloplasmin, chitinase-3-like protein 1, collagenpeptidase, complementC3 (including C3dg and its cleavage at the thioester site between aminoacids 1010-1013), complement factor H-related protein 1, connectivetissue growth factor, collagen IV, collagen VI, collagen XIV, 72 kDatype IV collagenase, corticosteroid-binding globulin, creatinine,dickkopf-1, epithelial cell adhesion molecule, fibrinogen gamma chain,fibronectin type III domain-containing protein 5, ficolin-1, full bloodcount, galactosylhydroxylysyl-glucosyltransferase, gamma-glutamyltranspeptidase, gelatinase B, gelsolin, glypican-3, golgi membraneprotein 1, haptoglobin (including the beta chain at pH 5.46-5.49 withglycans which are mainly biantennary, both mono- or disialylated withhardly any tri- or tetra-antennary/sialylated structures and less sialicacid and more monosialylated structures than the other haptoglobinisoforms of lower pH), haptoglobin-related protein, hemoglobin A1c,hemopexin, high density lipoprotein (HDL), hyaluronic acid,immunoglobulin J chain, inter-alpha-trypsin inhibitor (H1, H2, H5, H6),intracellular adhesion molecule 1, irisin, laminin subunitalpha/beta/gamma (including laminin P1-fragment), lecithin cholesterolacyl-transferase, liver function tests, low density lipoprotein (LDL),lysylhydroxylase, lysyloxidase, matrix metalloproteinases (MMP-1 toMMP-28), metalloproteinase inhibitors (TIMP1, TIMP2, TIMP3, TIMP4),microfibril-associated protein 4, monoamine-oxidase,N-Acetyl-beta-d-glucosaminidase, osteopontin, peroxiredoxin-2,phosphatidylcholine-sterol acyltransferase, pigment epithelium-derivedfactor, platelet count, platelet-derived growth factor, procollagen typeI (including N-terminal propeptide and C-terminal propeptide),procollagen type III (including intact procollagen, N-terminalpropeptide, C-terminal propeptide, complete propeptide Col 1-3),globular domain of propeptide Col-1), prolylhydroxylase, 14-3-3 proteinzeta/delta, protein AMBP, prothrombin (index/prothrombin time/INRratio), resistin, retinol-binding protein 4, sal-like protein 4, serumparaoxonase/arylesterase 1, sex hormone-binding globulin, SNC73,talin-1, tenascin, thrombospondin-2, thrombospondin-3, thrombospondin-4,thrombospondin-5, transferrin, transforming growth factor alpha (TGFalpha), transforming growth factor beta-1 (TGF beta-1), tropomyosin(1-4), tumor necrosis factor (TNF alpha), type IV collagen (includingNC1-fragment C-terminal crosslinking domain PIVP and 7S domain/7Scollagen), type VI collagen, undulin, vascular cell adhesion moleculeand/or vitronectin.

EXAMPLES

Differentially Expressed Proteins Established when Comparing Plasma fromPatients with Different Stages of NAFLD

The inventors have discovered that various proteins are differentiallyexpressed in human serum samples of NAFLD patients. The following groupswere compared: NAFL, NASH with no fibrosis F0, NASH with mild fibrosisF1 and NASH with advanced fibrosis F3. This discovery was achieved bycomparing these serum samples using two proteomics methods: Twodimensional polyacrylamide gel electrophoresis (2D-PAGE) and TMT-basedmass spectrometry.

Example 1—Two Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE)

To identify biomarkers for NAFLD, serum samples were analyzed using two2D-PAGE-based proteomics approaches. The following NAFLD stages wereanalyzed: NAFL, NASH with no fibrosis F0, NASH with mild fibrosis F1 andNASH with advanced fibrosis F3.

In the first approach, twelve highly abundant serum proteins weredepleted from all NAFLD samples by immunoprecipitation using the Top 12Abundant Protein Depletion Spin Column (Thermo Scientific, Loughborough,UK) according to the manufacturer's protocol. The 12 proteins which weredepleted were alpha 1-acid glycoprotein, alpha 1-antitrypsin, alpha2-macroglobin, albumin, apolipoprotein A-I, apolipoprotein A-II,fibrinogen, haptoglobin, IgA, IgG, IgM and transferrin. Fifty microgramsof the depleted serum proteins were separated by charge using a 7 cm pH3-11 non-linear gradient in the first dimension of the gel followed bymolecular weight (size) in the second dimension using a 4-12% (w/v)SDS-PAGE gradient. Electrophoresis, fluorescent staining and scanning ofgels were performed as described by Gangadharan et al., (2007), ClinChem, 53, 1792.

In the second approach, two milligrams of undepleted serum proteins wereseparated by charge using a pH 3-5.6 non-linear gradient in the firstdimension o followed by molecular weight in the second dimension using a9-16% (w/v) SDS-PAGE gradient. Electrophoresis, fluorescent staining andscanning of gels were performed as described by Gangadharan et al.,(2012), PLoS ONE, 7, e39603; Gangadharan et al., (2011), Nature ProtocolExchange, doi:10.1038/protex.2011.261 and Gangadharan et al., (2011), JProteome Res, 10, 2643.

Example 2—Differential Image Analysis and Protein Identification

The two dimensional array of spots generated were compared among thedifferent NAFLD stages by computer-aided image analysis. Scanned imagesof all 2D-PAGE gels were analyzed by computer-aided image analysis usingthe Progenesis SameSpots software (Nonlinear Dynamics Limited,Newcastle, UK). Images were processed in an automated linear workflow,including gel alignment, spot detection, spot splitting and statisticalanalysis. Spot detection produced a complete data set as all gelscontain the same number of spots, each matched to its corresponding spoton all gels. There were no missing values allowing valid statisticalanalysis to be applied. Only differentially expressed changes that were1.5-fold or more different and also had ANOVA p-value ≤0.05 for 95%confidence were considered to be statistically significant. All changesshown on the list were visualized and confirmed across all gels. A totalof 15 differentially expressed features were observed using the pH 3-11gels with depleted serum. A total of 38 differentially expressedfeatures were observed using the pH 3-5.6 gels with undepleted serum.Overall 11 proteins were identified in features increasing with NAFLDseverity and 35 proteins were identified in features decreasing withNAFLD severity (i.e. a total of 46 proteins). Examples of differentiallyexpressed features are shown in FIG. 1. These differentially expressedfeatures were excised from the gels, the proteins in the gel pieces weredigested with trypsin, and the peptides were analyzed by massspectrometry to identify the biomarkers essentially as described byGangadharan et al., (2007), Clin. Chem., 53, 1792 and Gangadharan etal., (2012), PLoS ONE, 7, e39603.

The proteins in the NAFLD serum samples were separated by SDS-PAGEfollowed by Western blotting using antibodies against the novelbiomarkers. Apolipoprotein F and kininogen-1 were shown to decreaseacross all NAFLD stages. Apolipoprotein D was higher in healthy controlscompared to all stages of NAFLD suggesting that it has the potential tobe an early NAFLD marker and capable of differentiating healthy controlsfrom patients with NAFL. Apolipoprotein M levels appeared to beconsistent in healthy controls and all NAFLD stages except for NASH F3suggesting that this is an advanced fibrosis biomarker. The Westernblots for these four NAFLD biomarkers are shown in FIG. 2 and these fourbiomarkers were considered as particularly preferred biomarkers out ofthe original list of 46 proteins initially identified by 2D-PAGE.

Example 3—TMT-Based Mass Spectrometry

Two serum samples from each of the following five stages wereanalyzed: 1) healthy individuals, 2) NAFL, 3) NASH with no fibrosis F0,4) NASH with mild fibrosis F1 and 5) NASH with advanced fibrosis F3. Theproteins in these ten samples were digested with trypsin and the trypticpeptides were labelled with an isobaric isotopically labelled tandemmass tag (TMT) according to the manufacturer's protocol (LifeTechnologies). All ten samples with TMT labelled peptides were mixed andanalyzed by LC-MS/MS using the Dionex Ultimate 3000 UHPLC and Thermo QExactive hybrid quadrupole-Orbitrap mass spectrometer. Using this methoda total of 54 differentially expressed serum proteins were observed.Examples of differentially expressed proteins based on relativequantitation TMT data are shown in FIG. 3.

All 54 differentially expressed proteins were targeted by PRM, withthree peptides for each protein wherever possible, using tryptic digestsof the serum samples. This validation check helped to identify which ofthe 54 potential NAFLD biomarkers were the most promising. Of the 54proteins, 8 proteins not previously identified in Example 2 were foundto be particularly promising biomarkers (thus giving a total of 12particularly promising biomarkers including the 4 listed in Example 2).From the list of 8 proteins, ficolin-2, adiponectin, alpha-1-acidglycoprotein 2 and leucine-rich alpha-2-glycoprotein all showed adecrease across NAFLD stages whereas lipopolysaccharide-binding protein,thrombospondin-1, IgG Fc-binding protein and cystatin-c showed anincrease across NAFLD stages. The PRM data for these 8 NAFLD biomarkersalong with apolipoprotein D are shown in FIG. 4.

Example 4—NAFLD and/or Fibrosis Scoring Scale

A NAFLD and/or fibrosis scoring scale for each of the novel biomarkerscan be formulated. The average concentration of these biomarkers incapillary blood, venous blood, arterial blood, serum, plasma or otherbodily fluid over the various stages of NAFLD and/or liver fibrosis isdetermined. In NAFLD, the Kleiner-Brunt scale of 0 to 4 is presentlyused to assess liver fibrosis in the clinic where 0 represents nofibrosis, 1-3 represent the intermediate stages of fibrosis inincreasing severity from mild to moderate/severe and 4 is cirrhosis(Kleiner, (2005), Hepatology, 41, 1313). Fibrosis can also be stages onother similar scales from 0 to 4 (such as Metavir, Knodell) or 0 to 6where 0 represents no fibrosis, 1-5 represent the intermediate stages offibrosis in increasing severity from mild to moderate/severe and 6 iscirrhosis (Ishak, (1995), J Hepatol, 22, 696). By determining theconcentration ranges of the novel biomarkers across these stages, asimilar mathematical scoring algorithm as in Kleiner-Brunt or similarcan be assigned to help provide information on staging and prognosis.The additive results from the scores of more than one of the novelbiomarkers may give a more reliable indication of the degree of fibrosisrather than examining individual biomarkers. The scoring method may notnecessarily follow existing scores such as Kleiner-Brunt, Ishak,Metavir, Knodell or the like and may be a novel scoring scale to helpdetermine NAFLD and/or fibrosis stage and to determine if treatment isnecessary.

Example 5—Quantitation by Mass Spectrometry

An antibody-free approach using mass spectrometry may be used todetermine NAFLD and/or fibrosis stage by detecting and/or quantifyingone or more biomarkers of interest. The proteins in the biologicalsample (either capillary blood, venous blood, arterial blood, serum,plasma or other bodily fluid) are usually denatured with urea or thelike. Proteins can optionally be reduced (using dithiothreitol,tris(2-carboxyethyl)phosphine, beta mercaptoethanol or the like) andalkylated (using iodoacetamide or the like) and these steps are usuallycarried out prior to digestion although they can be carried out postdigestion. Proteins are digested with an enzyme such as trypsin,chymotrypsin or the like. In the case of trypsin, this also includesimmobilized trypsin for higher throughput digestion such as Flash digestfrom Perfinity Biosciences (also called SMART Digest from ThermoScientific). Protein digestion can be carried out using in-solutiondigestion, Filter Aided Sample Preparation (FASP), in-gel digestion(where the sample is either run into an SDS-PAGE gel, optionally stainedand excised for digestion or is cast in a gel block of polyacrylamidebefore digestion), immobilized enzyme digestion (such as Flash Digestfrom Perfinity Biosciences or SMART Digest from Thermo FisherScientific) or the like. Isotopically labeled peptide standards can bespiked into the biological sample either before or after digestion andthese include synthetic peptides, IGNIS peptides, PEPotec peptides, AQUApeptides, SpikeTide peptides, Protein Epitope Signature Tag (QPrEST)peptides, microwave-assisted solid phase synthesized peptides,concatenated signature peptides encoded by QconCAT genes or the like.

The protein cleavage sites for different enzymes such as trypsin areknown and so the biomarkers are digested in-silico either manually orusing a software (such as Skyline, PinPoint or the like) to helpdetermine suitable peptides for targeted quantitation. Typicallypeptides selected are longer than 7 amino acids, shorter than 25 aminoacids, not modified (such as glycosylation, phosphorylation or the like)and not containing a methionine although peptides outside this criteriashould not be entirely excluded. Online databases such as, but notlimited to, PeptideAtlas and the Global Proteome Machine database(gpmdb), help to determine the commonly observed peptides for theproteins of interest. MRMaid can be used to help design assays for massspectrometry based targeted quantitation by suggesting peptides and MS2ions to monitor based on experimental spectra from the PRIDE database.Protein BLAST is used to check the uniqueness of the selected peptide.If a peptide is found to be not unique, then another peptide within thebiomarker sequence would be selected.

Prior to mass spectrometry, digested peptides can be fractionated usingphosphopeptide enrichment, glycopeptide enrichment, high pH reversedphase fractionation or the like. Peptides can be separated by reversedphase liquid chromatography, hydrophilic interaction liquidchromatography (HILIC), ion-exchange chromatography, isoelectricfocusing or the like.

SRM/MRM is performed using a triple quadrupole mass spectrometer whichhas three quadrupoles which include instruments such as the Thermo TSQQuantiva, Waters Xevo TQ-S, Agilent 6495, Bruker EVOQ, ABSciex 3500,Shimadzu 8050 or the like. PRM is performed using a hybridquadrupole-Orbitrap mass spectrometer such as a Thermo Q Exactive andits variants or a Thermo Fusion and its variants. MRM3 is performedusing a QTRAP mass spectrometer such as the ABSciex QTRAP 6500. The areaunder the curve (AUC) of the fragment ions can be used to relativelyquantify the levels of the proteins of interest. Known amounts ofsynthetic pure heavy-labelled peptides, such as AQUA peptides or thelike, with the same sequence as the preselected peptides can be used toestablish a calibration curve to help determine the absoluteconcentration of the proteins. Alternatively absolute quantitation canbe carried out using the HeavyPeptide IGNIS Prime Custom PeptideQuantitation Kit from Life Technologies.

The inventors also propose that any biomarkers which cleave or have aregion of interest could be targeted by mass spectrometry using peptidestandards covering these cleavage regions or regions of interest to beused in a mass spectrometry assay.

Example 6—Immunoassay

An immunoassay may be performed to assess the levels of biomarker suchas enzyme linked immunosorbent assay (ELISA), radio-immunoassay, proteindot blot, Western blot, turbidimetry, nephelometry and the like.

In the cases of an ELISA, the assay can be performed in a 96-well plate.One option is to use the non-competitive one-site binding ELISA. In thisassay, capillary blood, venous blood, arterial blood, serum, plasma orother bodily fluid along with known concentrations of antigen areprepared in a buffer such as a bicarbonate buffer, added to the 96-wellplate and incubated over a set period such as overnight at 4° C. Thewells are then washed with a solution such as phosphate buffered saline(PBS) and Tween (PBS-T) or the like followed by blocking with a PBSsolution containing bovine serum albumin or the like. After anincubation period (such as 37° C. for 1 hour), a primary antibodydirected against the antigen (in this example, the biomarker ofinterest) is added, the plate incubated for a period (such as 37° C. for1 hour) and then washed with PBS-T or the like. A conjugated secondaryantibody (such as horseradish peroxidase), directed against the animalorigin of the primary antibody, is then added, and the plate incubatedfor a period (such as 37° C. for 1 hour) followed by washes with PBS-Tor the like. Finally, a substrate (e.g. a peroxidase substrate such as2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid, ABTS) is added toeach well and the absorbance read on a plate reader (e.g. at 405 nmafter 30 minutes for ABTS).

Alternatively, a sandwich ELISA can be used. In this type of assay, oneantibody is bound to the bottom of a plate well. The antigen, in thiscase the biomarker protein, is added and unbound products are removed bywashing. A second, labeled antibody that binds to the antigen is thenadded. The amount of bound secondary antibody is quantified, usuallycolorimetrically. In addition to the novel biomarkers, the inventorspropose that any biomarkers which cleave or have a region of interest,then antibodies could be raised which target these cleavage regions orregions of interest to be used in an immunoassay.

The invention claimed is:
 1. A method of treating non-alcoholic fattyliver disease (NAFLD), the method comprising: diagnosing NAFLD in anindividual, by a method comprising diagnosing, prognosing or monitoringor staging progression of NAFLD in the individual, the method comprisingdetecting and quantifying one or more biomarkers in a biological sampleobtained from the individual, wherein the one or more biomarkers areselected from the group consisting of apolipoprotein F, ficolin-2,apolipoprotein D, apolipoprotein M, IgG Fc-binding protein, cystatin-c,and leucine-rich alpha-2-glycoprotein, wherein the method comprisescomparing the level of the one or more biomarkers to a control sample orreference sample/level and determining whether the level of the one ormore biomarkers is indicative of progression of NAFLD in the individual,wherein: a decreasing level of apolipoprotein F, ficolin-2,apolipoprotein D, apolipoprotein M and/or leucine-richalpha-2-glycoprotein, as compared with a control sample or referencesample/level, indicates increasing severity of NAFLD as it progresses;and/or an increasing level of IgG Fc-binding protein, or cystatin-c, ascompared with a control sample or reference sample/level, indicatesincreasing severity of NAFLD as it progresses, and thereby diagnosing,prognosing or monitoring or staging the progression of NAFLD; andwherein the method further comprises: administering to the individual anagent or carrying out a treatment regimen effective to treat theprogression of NALFD in the individual.
 2. The method of claim 1,wherein the method comprises staging the progression of NAFLD, whereinthe NAFLD is staged as non-alcoholic fatty liver, non-alcoholicsteatohepatitis, hepatic fibrosis or cirrhosis.
 3. The method of claim1, wherein the NAFLD is staged as non-alcoholic fatty liver.
 4. Themethod of claim 1, wherein the one or more biomarkers are selected fromthe group consisting of apolipoprotein F, ficolin-2, apolipoprotein D,apolipoprotein M, IgG Fc-binding protein, and leucine-richalpha-2-glycoprotein.
 5. The method of claim 1, wherein two or more,three or more or four or more biomarkers are selected from the groupconsisting of apolipoprotein F, ficolin-2, apolipoprotein D,apolipoprotein M, IgG Fc-binding protein, cystatin-c, and leucine-richalpha-2-glycoprotein.
 6. The method of claim 1, comprising detecting andquantifying two or all three of the biomarkers apolipoprotein F,ficolin-2, and apolipoprotein D.
 7. The method of claim 1, furthercomprising detecting and quantifying thrombospondin-1, wherein anincreasing level of thrombospondin, as compared with a control sample orreference sample/level, indicates increasing severity of NAFLD as itprogresses through the stages, optionally wherein the method comprisesdetecting and quantitating one or more peptides selected fromIEDANLIPPVPDDK (SEQ ID NO: 23), GFLLLASLR (SEQ ID NO: 24) and/orAGTLDLSLTVQGK (SEQ ID NO: 25).
 8. The method of claim 1 furthercomprising detecting and quantifying: (i) adiponectin; (ii) keratin typeI cytoskeletal 18 and optionally its cleavage between amino acids397/398; and/or (iii) cleavage products of a protein, selected from thegroup consisting of kininogen-1, keratin type I cytoskeletal 18 andangiotensinogen.
 9. The method of claim 1, comprising detecting andquantifying the biomarker apolipoprotein F and one or more biomarkersselected from the group consisting of ficolin-2 and apolipoprotein D.10. The method of claim 1, wherein the biological sample is blood, serumor plasma.
 11. The method of claim 1, wherein the individual is alreadydiagnosed with NAFLD.
 12. The method of claim 1, wherein the sample isanalysed using a mass spectrometry method which detects and quantitatesthe one or more peptides derived from one or more biomarkers selectedfrom the group consisting of apolipoprotein F, ficolin 2, apolipoproteinD, apolipoprotein M, IgG Fc-binding protein, cystatin-c, leucine-richalpha-2-glycoprotein.
 13. The method of claim 12, wherein the massspectrometry method is Parallel-Reaction Monitoring (PRM), SelectedReaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), ormulti-stage fragmentation in MRM-cubed (MRM3).
 14. The method of claim1, wherein the sample is analysed using an immunoassay.
 15. The methodof claim 14, wherein the immunoassay is ELISA, a radioimmunoassay, dotblot, Western blot, turbidimetry or nephelometry.
 16. The method ofclaim 1, wherein the biomarker cystatin-c is detected and quantitated bya peptide ALDFAVGEYNK (SEQ ID No: 32).
 17. The method of claim 1,wherein the one or more biomarkers are selected from the groupconsisting of apolipoprotein F, ficolin-2, apolipoprotein D,apolipoprotein M, IgG Fc-binding protein and leucine-richalpha-2-glycoprotein, and the method comprises detecting andquantitating one or more peptides selected from SLPTEDCENEK (SEQ ID NO:1), SGVQQLIQYYQDQK (SEQ ID NO: 2), DANISQPETTK (SEQ ID NO: 3) and/orSYDLDPGAGSLEI (SEQ ID NO: 4), wherein the biomarker is apolipoprotein F;VDGSVDFYR (SEQ ID NO: 20), LGEFWLGNDNIHALTAQGTSELR (SEQ ID NO: 21)and/or NCHVSNLNGR (SEQ ID NO: 22), wherein the biomarker is ficolin-2;VLNQELR (SEQ ID NO: 42), NPNLPPETVDSLK (SEQ ID NO: 43) and/orNILTSNNIDVK (SEQ ID NO: 44), wherein the biomarker is apolipoprotein D;YDLAFVVASQATK (SEQ ID NO: 29), LDSLVAQQLQSK (SEQ ID NO: 30) and/orGATTSPGVYELSSR (SEQ ID NO: 31), wherein the biomarker is IgG Fc-bindingprotein; and/or ALGHLDLSGNR (SEQ ID NO: 39), VAAGAFQGLR (SEQ ID NO: 40)and/or GQTLLAVAK (SEQ ID NO: 41), wherein the biomarker is leucine-richalpha-2-glycoprotein.
 18. The method of claim 12, which comprisesdetecting and quantitating one or more unique peptides derived from saidone or more biomarkers.
 19. The method of claim 12, wherein a saidpeptide derived from a said biomarker is of 7 to 25 amino acids inlength.
 20. The method of claim 12, which comprises use of one or moreisotopically labelled synthetic peptides having the same sequence as theone or more peptides derived from said one or more biomarkers, forquantification of the level of said peptides.
 21. The method of claim12, wherein the peptides are derived by digestion of the biologicalsample with a protease.
 22. The method of claim 1, further comprisingdetecting and quantifying the biomarker alpha-1 acid glycoprotein 2,wherein a decreasing level of alpha-1-acid glycoprotein 2 as comparedwith a control sample or reference sample/level, indicates increasingseverity of NAFLD as it progresses through the stages.
 23. The method ofclaim 1, further comprising detecting and quantifying the biomarkerkininogen-1, wherein a decreasing level of kininogen-1, as compared witha control sample or reference sample/level, indicates increasingseverity of NAFLD as it progresses through the stages, optionallywherein the method comprises detecting and quantitating one or morepeptides selected from ISLMKRPPGFSPFR (SEQ ID NO: 8), RPPGFSPFR (SEQ IDNO: 9) and/or KRPPGFSPFR (SEQ ID NO: 10).
 24. The method of claim 1,further comprising detecting and quantifying the biomarkerlipopolysaccharide-binding protein, wherein an increasing level oflipopolysaccharide-binding protein, as compared with a control sample orreference sample/level, indicates increasing severity of NAFLD as itprogresses through the stages, optionally wherein the method comprisesdetecting and quantitating one or more peptides selected from SFRPFVPR(SEQ ID NO: 33), ITGFLKPGK (SEQ ID NO: 34) and/or VQLYDLGLQIHK (SEQ IDNO: 35).